Inhibition of Angiogenesis by Adenovirus-Mediated sFlt-1 Expression in a Rat Model of Corneal Neovascularization

Abstract

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy

“…In our cauteryinduced study of corneal NV, injection with Ad.sflt achieved a 70% decrease in the number of vascularised corneas, 27 while injection with AAV-CMV.sflt reduced NV Gene Therapy only by 36%. This lower efficacy of AAV-CMV.sflt was probably due to a combination of lower titer and lower transduction efficiency of AAV in the anterior chamber, particularly in the corneal endothelium from which secreted sFlt-1 proteins have closer access to the cautery lesion.…”

“…47 The preparation of Ad.sflt has been described. 27 AAV vector titers were determined using a DNase-resistant physical particle assay. 47 Titers for viruses were 4 × 10 11 particles/ml of AAV-CMV.sflt, 8 × 10 11 particles/ml of AAV-CMV.gfp and 10 11 plaqueforming units (p.f.u.…”

“…Both models have been shown to induce VEGF expression resulting in extensive blood vessel growth in the normally avascular cornea and subretinal space, respectively. 22,26,27 Based upon the proven efficacy of soluble VEGF receptor 1, sFlt-1, as a potent anti-angiogenic agent, [28][29][30] we selected sFlt-1 as our therapeutic gene and investigated the efficacy of AAV-mediated delivery and expression of this protein as SGT against global corneal NV and choroidal NV.…”

Discordant effects of a soluble VEGF receptor on wound healing and angiogenesis

“…11 Similar antiangiogenic properties were demonstrated in the corneal micropocket assay in which Flk-1 and Flt-1 strongly inhibited corneal neovascularization. 11,16 Based on these findings, we hypothesized that gene transfer with soluble VEGF receptors might also impair wound healing. Of note, animals in this study were injected with the same systemic dose (10 9 PFU) of the respective adenoviruses that were found to be highly effective in suppressing tumor growth and corneal neovascularization in the above-mentioned studies.…”

“…[11][12][13][14][15] Furthermore, in corneal micropocket assays in rodents, gene delivery significantly attenuated neovascularization induced by sustained release VEGF pellets. 11,16 Wound healing is a complex, dynamic process in which angiogenesis has been hypothesized to play an integral part since the formation of new blood vessels is a necessary step that allows a variety of mediators and regulators to reach the center of the healing process. 17 VEGF appears to play a crucial role during the proliferative phase of wound healing.…”